Field of the Invention
The present invention relates to a method of making measurements on quadrupolar nuclei which can be observed by NMR spectroscopy.
Description of Related Art
A nuclear magnetic resonance (NMR) spectrometer is an analytical instrument for detecting signals of atomic nuclei having spin magnetic moments by applying a static magnetic field to these atomic nuclei to induce a Larmor precession in the spin magnetic moments and irradiating the atomic nuclei with RF waves having the same frequency as the Larmor precession so as to produce a resonance.
There are 120 nuclear species, in total, that can be observed by NMR spectroscopy, i.e., nuclear species having nuclear spins. A number of integer spin nuclei are included among them. Important nuclear species such as 14N nucleus are included among them. First, the resolution of integer spin S is described.
An integer spin S has two features. One is that there exist quadrupolar interactions which are present in nuclear spins with I>½. Quadrupolar interactions are always present in integer spins S with I>½.
Such quadrupolar interactions might be quite large, and some of them exceed tens of MHz or more. Furthermore, the degree of broadening of a spectrum due to a quadrupolar interaction varies depending on the relative orientation between spin and magnetic field. Therefore, in a powdered sample, there are quadrupolar interactions of various magnitudes.
Another feature of integer spin S is that every single quantum transition is affected by first-order quadrupolar interactions. In normal NMR measurements, single quantum transitions are observed and so it can also be said that normal NMR measurements of integer spins S are affected by first-order quadrupolar interactions. That is, in an NMR spectrum of integer spins S, resonance spins appear at positions reflecting the magnitudes of quadrupolar interactions.
Accordingly, in a spectrum of a powdered sample with integer spin S, resonance lines appear at various positions reflecting the distribution of the sample orientation, leading to spectral broadening. Taking 14N nucleus with spin S=1 as an example, the effects of quadrupolar interactions are described below by referring to FIG. 1.
If energy splitting is only Zeeman splitting, and if an NMR measurement is made, a single sharp peak appears at position νL. However, in the case of integer spin S, energy levels of ±1 vary due to first-order quadrupolar interactions. As a result, two sharp split peaks appear at positions νL±νQ.
In the case of powdered sample, the values of νQ are distributed due to the spin orientation. Peaks appear at various positions and overlap each other, producing a broad signal. This broad signal is referred to as a first-order quadrupolar powder pattern. In the case of 14N nucleus, quadrupolar interactions typically have magnitudes on the order of MHz. Corresponding first-order powder patterns exhibit linewidths on the order of MHz.
In this way, an NMR spectrum of integer spin S is affected by quadrupolar interactions. This gives rise to broadening of signal. Furthermore, quadrupolar interactions are quite large and, therefore, second-order quadrupolar broadening corresponding to second-order perturbation terms also takes place. Second-order quadrupolar interactions are also included in the energy levels of FIG. 2. Where up to second-order terms are taken into consideration, an NMR signal produces peaks at positions νL±νQ(1)+νQ(2).
Both of νQ(1) and νQ(2) vary due to relative orientation between spins and magnetic field. In the case of a powdered sample, therefore, an NMR signal is observed as a superposition of first-order and second-order powder patterns. Second-order broadening is vastly smaller than first-order broadening.
The following methods are available to remove broadening.
Related Art 1: Magic-Angle Sample Spinning (MAS)
Application of a pulsed RF magnetic field having a frequency close to the Larmor frequency of integer spin S permits excitation and observation of single quantum transitions. Furthermore, the first-order pattern of integer spin S can be averaged by applying magic-angle sample spinning (MAS) to the sample. MAS is a technique for spinning a sample at high speed about an axis tilted by a magic angle relative to a magnetic field, and is often used in solid sample NMR techniques.
The obtained NMR spectrum is observed to be split into a group of spinning sidebands (SSBs) in a comb-like form (see FIG. 3). The individual peaks are somewhat more sharpened by MAS even in the case of second-order powder patterns and thus are high-resolution peaks. Second-order powder patterns remain but their magnitudes are extremely smaller than first-order powder patterns. Therefore, an overwhelmingly great increase in resolution is achieved. One problem is that there are quite many SSBs. That is, the signal intensity is dispersed and the sensitivity is low. Another problem is that if any slight deviation from the magic angle occurs, powder patterns are erased incompletely to thereby produce broadening, because first-order quadrupolar powder patterns are deleted by MAS.
Related Art 2: SQ-HMQC, SQ-HSQC under MAS
As described already in connection with related art 1, first-order quadrupolar broadening is eliminated by MAS and high-resolution measurements of NMR signals of integer spins S are enabled. However, there is the problem that the signal is split into a large number of SSBs, leading to a decrease in sensitivity.
Accordingly, a technique also employing indirect measurements using other nuclear species has been proposed. In particular, a spectrum of integer spin S is placed in an indirect observation dimension. Observations are made indirectly through spin I. Since single quantum transitions are selected for the integer spin S, the spectrum in the indirect observation dimension is quite close to spectra obtained by the related art 1.
One example of integer spin S indirectly observed is illustrated in FIG. 4, where 14N nucleus with I=1 is taken as one example and I=½ is directly observed. The sample is measured under MAS. The measurement is similar to the HMQC technique except that the indirect observation dimension (t1-dimension) is synchronized with the sample spinning period. That is, the period of the t1-dimension is set to be an integral multiple of the sample spinning period τr, i.e., t1=nτr.
This technique is published in S. Cavadini et al., Journal of the American Chemical Society 128 (2006) 7706 and Z. Gan, Journal of the American Chemical Society 128 (2006) 6040 and constitutes U.S. Pat. No. 7,276,903. Pulses applied to integer spin S have a frequency close to the Larmor frequency of integer spin S. This technique has three features:
(1) Since the process is started from initial magnetization of a nucleus with I=½ that is greater than the initial magnetization of 14N nucleus, the NMR detection sensitivity is enhanced.
(2) Since the nucleus with I=½ of higher sensitivity (higher Larmor frequency) than 14N nucleus in the t2-dimension is observed, the NMR detection sensitivity is improved.
(3) Since the dimension of 14N nucleus (indirect measurement dimension: t1-dimension) is synchronized with the sample spinning period, all SSBs of 14N nucleus are observed to be overlapped at the center, thus improving the NMR detection sensitivity.
FIG. 5 illustrates the feature (3) above. Because a signal split into numerous SSBs is superimposed in a central peak, the NMR detection sensitivity is improved. As a result, first-order quadrupolar broadening is removed from the resulting NMR spectrum of 14N nucleus in the indirectly observed dimension and the effects of second-order quadrupoles remain.
Peak positions are determined by second-order quadrupolar shift and isotropic chemical shift, and the lineshape is determined by second-order quadrupolar powder pattern. In addition, it is known that third-order quadrupolar powder pattern affects the lineshape.
At first, magnetization transfer between spins I and S was effected by heteronuclear J coupling and heteronuclear residual dipolar splitting (RDS). Later, a method using heteronuclear dipolar coupling was also proposed (see Z. Gan et al., Chemical Physics Letters 435 (2007) 163).
As a modification of this technique, heteronuclear single quantum coherence (HSQC) for bisecting a 180-degree pulse at the center of spin I is also proposed as shown in FIG. 6 (see S. Cavadini et al., Journal of Magnetic Resonance 190 (2008) 160-164).
These techniques succeeded as high-sensitivity, high-resolution correlation NMR methods. However, there is the problem that measurements are quite sensitive to adjustment of the magic angle and that experimental adjustments are quite difficult to make. In this technique, first-order quadrupolar interactions are eliminated by MAS. Accordingly, only a slight deviation from the magic angle results in incomplete removal of first-order dipolar interactions. In particular, it is reported that even a deviation of 1/100 degree distorts the spectrum.
Related Art 3: DQ-HMQC and DQ-HSQC
A double-quantum (DQ) method has been proposed as a technique of solving the problem with adjustment of the magic angle, i.e., the problem with SQ (single quantum)-HMQC/HSQC methods. This method employs double quantum transitions in order to remove first-order quadrupolar interactions (see S. Cavadini et al., Journal of the American Chemical Society 128 (2006) 7706 and Z. Gan, Journal of the American Chemical Society 128 (2006) 6040).
Energy levels are shown in FIG. 7, where 14N nucleus with S=1 is taken as one example. Single quantum transitions include first-order quadrupolar interactions. On the other hand, double quantum transitions do not include first-order quadrupolar interactions. That is, if double quantum transitions can be observed, it is possible to eliminate first-order quadrupolar interactions.
Since the elimination of first-order quadrupolar interactions does not rely on MAS, a slight deviation from the magic angle does not greatly affect the spectrum. Because third-order quadrupolar interactions are simultaneously removed, the lineshape is determined only by second-order quadrupolar powder pattern.
No spinning sidebands appear and so the restriction t1=nτr is not imposed, unlike in SQ-HMQC/HSQC where the restriction is placed to superimpose spinning sidebands. That is, there is the advantage that no restrictions are imposed on the spectral width in the directly observed dimension but rather the width can be set at will.
DQ-HMQC/HSQC is observed with the same pulse sequence as for SQ-HMQC/HSQC. However, phase rotation is effected to select double quantum transitions of 14N nucleus in the t1-dimension. The double quantum transitions of 14N nucleus are caused by excitation near the Larmor frequency of 14N nucleus. DQ-HMQC/HSQC is not sensitive to the setting of the magic angle but permits NMR measurements to be made easily. However, there is the disadvantage that the sensitivity is low because of low double quantum excitation efficiency.
Related Art 4: Overtone NMR Spectroscopy
A method using RF pulses having a frequency that is double the Larmor frequency in order to directly excite double quantum transitions of integer spins has been proposed. This method is known as overtone NMR spectroscopy, and permits direct excitations and direct observation of double quantum transitions.
In recent years, it has been shown that overtone NMR spectroscopy can be performed under MAS. Although overtone NMR spectroscopy enables high-resolution measurements, high-sensitivity measurements cannot be always made because of low overtone excitations and detection efficiencies (see R. Tycko et al., Journal of Chemical Physics 86 (1987) 1761 and L. A. O'Dell et al., Chemical Physics Letters 514 (2011) 168).
As described so far, the four related art methods have the following problems. Related art method 1 (magic angle sample spinning) suffers from low sensitivity because a signal is split into numerous sample spinning sidebands. This method is sensitive to the magic angle. Any slight deviation leads to a deterioration in resolution. Related art method 2 (SQ-HMQC, SQ-HSQE under MAS) is sensitive to the magic angle. Any slight deviation leads to a deterioration in resolution. Related art method 3 (DQ-HMQC, DQ-HSQC) suffers from low sensitivity because of low double quantum excitation efficiencies. Related art method 4 (overtone NMR spectroscopy) suffers from low sensitivity because of low overtone excitations and low detection efficiencies.